A kind of phospholipase d mutant, recombinant genetic engineering bacteria and its preparation method and application

A technology of genetically engineered bacteria and mutants, which is applied in the field of genetic engineering of enzymes, can solve problems affecting the healthy development of the downstream enzymatic phospholipid modification industry, and achieve the effect of increasing the optimum reaction temperature and improving the vitality of mutants

Active Publication Date: 2020-07-28
SOUTH CHINA UNIV OF TECH
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no commercial PLD enzyme in China. Enzyme preparations mainly rely on imports. The high dependence on commercial enzyme sources has seriously affected the healthy development of my country's downstream enzymatic phospholipid modification industry.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A kind of phospholipase d mutant, recombinant genetic engineering bacteria and its preparation method and application
  • A kind of phospholipase d mutant, recombinant genetic engineering bacteria and its preparation method and application
  • A kind of phospholipase d mutant, recombinant genetic engineering bacteria and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1: Construction of phospholipase PLD-VH mutant expression vector and expression strain

[0023] (1) Referring to the complete amino acid sequence of Vibrio harveyi phospholipase PLD-VH (GenBank accession number: WP_005435673.1), the signal peptide sequence was analyzed by the signal peptide online analysis software Signal P, and the 1-24 Amino acids are deleted from the complete sequence to obtain the phospholipase PLD-VH mature peptide coding sequence (SEQ ID NO.1);

[0024] (2) According to the amino acid sequence obtained in (1), design the phospholipase PLD-VH gene coding sequence according to the codon preference of Escherichia coli, and its base sequence is shown in the accompanying drawing SEQ ID NO.4 (wild type). Nde I was introduced upstream of the sequence, and Xho I restriction site was introduced downstream, and the resulting phospholipase PLD-VH gene sequence was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.;

[0025] (3) The phospho...

Embodiment 2

[0043] Example 2: Wild-type PLD-VH and its mutant recombinant expression strain fermentation and recombinant protein purification

[0044] (1) Inoculate recombinant Escherichia coli PLD-VH wild-type and mutant expression strains in seed medium containing ampicillin 100 μg / mL (NaCl 10 g / L, peptone 10 g / L, yeast extract 5 g / L, pH 7.2 ~7.4), at 37°C, 200r / min shake flask culture to the logarithmic growth phase, as the seed solution;

[0045] (2) Inoculate the seed liquid described in (1) into the self-inducing liquid fermentation medium (enzyme hydrolyzed casein 10g / L, yeast extract 5g / L, glucose 0.5g / L, lactose 2g by 5% inoculum size) / L, glycerin 5g / L, disodium hydrogen phosphate 3.6g / L, potassium dihydrogen phosphate 3.4g / L, ammonium chloride 2.7g / L, sodium sulfate 0.7g / L, magnesium sulfate 1g / L, pH 7.2~ 7.4), shake the flask at 37°C and 200r / min until OD 600 = 0.6 ~ 0.8, and then induce culture at 20°C and 200r / min for 24h; (3) Centrifuge the fermentation broth obtained in ...

Embodiment 3

[0047] Embodiment 3: Phospholipase enzymatic property analysis

[0048] (1) Assay method of phospholipase activity

[0049] The activity of the wild-type phospholipase PLD-VH and mutant phospholipase was measured by a standard spectrophotometric method, and phosphatidyl-p-nitrophenol (P-pNP) was used as a reaction substrate. Take 480 μL of purified phospholipase enzyme solution, add 20 μL P-pNP solution, react at a certain temperature for an appropriate time, add 10% SDS to terminate the reaction, and measure the absorbance at 405 nm. Heat-inactivated enzyme protein solution was used as the reaction control group. Phospholipase activity is defined as: under specific temperature and pH conditions, the amount of enzyme required to release 1 μmol of p-nitrophenol per minute is a phospholipase activity unit. Represented by U. Calculate the specific enzyme activity (U / g).

[0050] (2) Determination of the optimal reaction temperature of the enzyme

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a phospholipase D mutant, recombinant genetically engineered bacteria and a preparation method and application thereof. The phospholipase D mutant is obtained by deleting 12 to47 amino acids at a C tail end on the basis of a parent sequence with the amino acid sequence of SEQ ID NO: 1. Two phospholipase D mutants are obtained and an experiment determines that the enzyme activity of the mutant 1 is improved by 3.7 times when being compared with that of a wild type mutant; meanwhile, the optimal reaction temperature is improved to 50 DEG C from original 45 DEG C; the enzyme activity of the mutant 2 is improved by 6 times when being compared with that of the wild type mutant; meanwhile, the optimal reaction temperature is improved to 60 DEG C from original 45 DEG C. According to the mutant disclosed by the invention, the optimal reaction temperature and the enzyme activity of the mutant are remarkably improved under the condition that the optimal pH (Potential ofHydrogen) is not changed; when the catalytic performance is improved, the industrial utilization value of the enzyme is further improved.

Description

technical field [0001] The invention belongs to the technical field of gene engineering of enzymes, and in particular relates to a phospholipase D mutant with significantly improved catalytic performance obtained by using molecular biology techniques and a preparation method for recombinant expression thereof in Escherichia coli. Background technique [0002] Phospholipase D (Phospholipase D, PLD) (EC 3.1.4.4) is a general term for a class of enzymes that catalyze the hydrolysis of phosphodiester bonds and base exchange reactions. PLD is distributed in many biological groups from bacteria to higher animals and plants. Its main physiological function in organisms is to participate in cell lipid metabolism, signal transduction and biofilm formation; It is an important tool enzyme for quality. Based on the unique base exchange activity of PLD, phospholipids can be modified to prepare high-purity single phospholipids, rare phospholipids and a series of functional phospholipids, ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/16C12N15/55C12N15/70C12N1/21C12R1/19
CPCC12N9/16C12N15/70C12Y301/04004
Inventor 王方华王永华吴宗泽杨博魏瑞霞
Owner SOUTH CHINA UNIV OF TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap